Mice that lack myosin Va (dilute) exhibit a diluted coat color (e.g. black mice become gray). This defect is due to a defect in the intracellular distribution of melanosomes (MSs) within melanocytes (MCs): instead of the normal accumulation of MSs at dendritic tips, the normal site of MS transfer from the MC to surrounding keratinocytes (KCs), MSs in dilute MCs are concentrated in the MCs central cytoplasm. Dilute mice that are also homozygous for a second mutation, dilute suppressor (dsu), exhibit a restoration of coat color (gray returns to black), but surprisingly without a restoration of MS distribution within MCs- they remain concentrated in the MCs central cytoplasm. Dsup (also known as melanoregulin) is a novel, highly charged, 22-kDa polypeptide, and it is the loss of expression of dsup that rescues the coat color of dilute mice (PNAS, 2005). We now find that dsup is exclusively a MS membrane protein in MCs. Membrane targeting is driven by myristoylation and multiple palmitoylation of dsup, and possibly by its interaction with cholesterol. FRAP analyses indicate that dsup rarely exits the MS membrane. Importantly, visualization in ear skin from WT, dilute and dilute/dsu mice of MC cell shape and MS distribution within MCs and KCs reveals that dsu rescues coat color without rescuing MS distribution within MCs by allowing the transfer of MSs concentrated in the center of dilute/dsu MCs to KCs surrounding the MC cell body. Moreover, examinations of the distribution of pigment in hair and ear skin from dsu mice that are WT for myosin Va are consistent with an augmentation of MS transfer to KCs when dsup alone is missing. Together, these results reconcile the apparent discrepancy between the animal and cellular phenotypes of dilute/dsu mice and suggest that this MS membrane protein serves as a negative regulator of MS transfer to KCs.[unreadable] Dictyostelium CARMIL (Dd CI) is a 116 kDa scaffold protein that binds myosin I (MI), Capping protein (CP) and the Arp2/3 complex at independent sites. Dd CI localizes along with MI, CP and Arp2/3 at sites of active actin assembly such as macropinocytic crowns and the leading edge of crawling cells, and Dd CI null cells exhibits defects in macropinocytosis and chemotaxis. Dd CI is composed of an N-terminal regulatory domain, a central LRR domain, and a C-terminal domain containing sequences resembling the verprolin/WH2 and acidic portions of WASP (VA domain; Arp2/3 interaction and weak activation) followed by a proline-rich domain (P; MI SH3 interaction) that terminates with CARMIL Homology domain-3 (CAH3). As in mammalian CARMILs, this 80-residue domain binds CP with low nM affinity and functions as a potent CP antagonist by binding free CP, thereby reducing its affinity for the barbed end 200 fold (sequestering activity), and by rapidly removing CP from the end of capped actin filaments (uncapping activity). We now find that the over expression (OE) of full length (FL) Dd CI results in cells with excess filopodia, a phenotype reminiscent of vertebrate cells following CP knock down (Cell, 2002). Consistent with previous evidence that the anti-CP activities of FL Dd CI are suppressed by intramolecular folding, an N-terminally truncated version of Dd CI (&#8710;NT Dd CI) is much more potent than FL Dd CI at inducing more and longer filopodia. Surprisingly, however, this effect persists when the CAH3 domain of &#8710;NT Dd CI is either mutated or deleted (although the induction of cortical actin patches/bumps is lost). OE of various portions of Dd CI (LRR-VAP, VAP, VA, AP, etc) argues that the induction of filopodia is due to the OE of the Dd CIs V/WH2-like sequence. Current efforts are directed at determining whether the filopodial phenotype is downstream of CARMIL-dependent effects on the activity/localization of the Arp2/3 complex.